NMDA receptors are multiprotein tetrameric complexes, which are composed of two NR1 subunits and two NR2A-2D subunits that form the ion channel for positive ions (Nature 438, 185-192 (2005)).
Glutamate is the major excitatory neurotransmitter in the central nervous system of mammals. Responses of the post synaptic neuron are generated during synaptic transmission via ionotropic and metabotropic glutamate receptors. N-Methyl-D-aspartate receptors (NMDA), AMPA and kainite receptors belong to the family of ionotropic glutamate receptors.
Although current evidence suggests the role of different subtypes of glutamate receptors in glutamate induced excitotoxicity, ionotropic receptors are considered to be a key player in these processes. Activation of ionotropic glutamate receptors leads to changes in intracellular concentration of ions, especially calcium and sodium. Toxicity of higher levels of glutamate is generally associated with an increase in intracellular Ca2+ levels. Currently, it is relatively well established that there is a direct relationship between the excessive influx of calcium into the cells and glutamate-induced neuronal damage. Glutamate-induced pathological increase in intracellular calcium is attributed to prolonged activation of ionotropic glutamate receptors. Increases in intracellular calcium may trigger a cascade of neurotoxicity.
A number of preclinical studies have documented striking ability of NMDA antagonists to prevent an excessive action of glutamate on nerve cells and thereby reduce the impairment of the function of CNS. However, from the clinical point of view their neuroprotective potential is small. Due to the fact that NMDA receptors are one of the most widespread types of receptors in the CNS, their administration lead usually to a number of serious side effects (e.g. distortion, induction of motoric psychoses of the schizophrenic type, etc.).
On the other hand, a great variety of NMDA receptors and their different distribution at synapses and in the brain and various functional states of this receptor offers great possibility of seeking for agents that selectively affect only a specific group of NMDA receptors and thereby reduce the occurrence of unanticipated and undesirable effects while maintaining the neuroprotective activity (Pharmacol. Rev. 51, 7-61 (1999); Semin. Cell Dev. Biol. 17, 592-604 (2006); Top. Med. Chem. 6, 749-770 (2006); Anesth. Analg. 97, 1108-1116 (2003); Curr. Opin. Pharmacol. 6, 53-60 (2006); Curr. Opin. Investig. Drugs 4, 826-832 (2003).
Previous results showed that naturally occurring 3alpha,5beta-pregnanolone sulfate affects the activity of NMDA receptors by the use-dependent manner. Due to this mechanism of action, pregnanolone sulfate has pronounced inhibitory action on NMDA receptors tonically activated by glutamate than phasically activated NMDA receptors during synaptic transmission. The activation of tonically activated extrasynaptic NMDA receptors is essential for the excitotoxic action of glutamate (J. Neurosci. 25, 8439-50 (2005)).
Therefore, we have started a development and testing of novel NMDA antagonists derived from neurosteroids. These newly synthesized compounds exhibit affinity for extrasynaptic NMDA receptors. Moreover, our previous electrophysiological studies have shown that this type of compounds binds only to the long-term opened-NMDA receptors. The supposed mechanism of the neuroprotective effect is blocking of excessive penetration of calcium into cells through the open NMDA receptors. As these compounds do not have affinity to other types of NMDA receptors, it is believed that they would minimally affect the signal transmission between neurons.
In the last decade, the biomedical research has been focused on research of the role of neurosteroids in the pathophysiology of many neuropsychiatric disorders and to assess therapeutic potential of these compounds. Mechanism of action of neurosteroids is associated with their activity on the NMDA and GABA receptors. Experimental studies with animal models suggest potential of neurosteroids to treat a variety of central nervous disorders, particularly neurodegenerative diseases, multiple sclerosis, affective disorders, alcoholism, pain, insomnia or schizophrenia (J. Pharm. Exp. Ther. 116, 1-6 (2007); J. Pharm. Exp. Ther. 293, 747 (2000)).
Neurosteroids play a crucial role in the regulation of stress and the related CNS disorders. The level of neurosteroids temporarily after exposure to stress increases, as it is an adaptive mechanism. On contrary, experimental models of chronic stress and depression on laboratory rodents show long-term reduced concentration of neurosteroids in the brain and in plasma, due to their reduced biosynthesis.
Similar findings are found in patients suffering from depression or premenstrual syndrome. These findings point to, a violation of homeostatic mechanisms in the CNS of neuropsychiatric disorders related to stress.
Among well-known neurosteroids belong pregnenolone, progesterone, dehydroepiandrosterone (DHEA) and its reduced metabolite, and sulfate esters. The regulation of the synthesis of neurosteroids in the CNS is not well known, but it is generally believed that the crutial is interaction of various types of cells. For example, progesterone synthesis by Schwann cells in peripheral nerves is regulated by diffuse signals from neurons.
Neurotrophic and neuroprotective effects of neurosteroids were shown both in cell cultures and by in vivo experiments. Progesterone plays an important role in neurological recovery from traumatic brain injury and spinal cord through mechanisms involving protection against excitotoxic cell damage, lipid peroxidation and induction of specific enzymes. For example, after spinal cord transection in rats progesterone increases the number of astrocytes expressing NO synthase just above and below the site of transection.
Neurosteroids thus significantly modulate the function of membrane receptors for neurotransmitters, in particular the GABAA receptor, NMDA receptor, and sigmal receptors. These mechanisms are responsible for psychopharmacological effects of steroids and partly explain their anticonvulsant, anxiolytic, sedative and neuroprotective effects as well as their influence on learning and memory processes.
For instance, pregnanolone sulfate was shown to be capable of reversing cognitive deficit in aged animals and exerting a protective effect on memory in several amnesia models of amnesia. Current studies have demonstrated direct effect of neurosteroids on intracellular receptors. Despite absence of direct evidence for binding of neurosteroids to corticoid receptors, they may obviously modulate their function indirectly, by interaction with protein kinases C and A, MAP-kinase or CaMKII. Moreover, pregnanolone and pregnanolone sulfate were shown to affect microtubule-associated proteins and increase the rate of microtubule polymeration, which may in turn affect neuronal plasticity. These newly described neurosteroid effects are still poorly understood, however, it can be assumed that they affect neuroprotectivity.
Sulfated and thus amphiphilic steroid compounds generally do not penetrate the blood-brain barrier, but it was demonstrated that intravenously administered pregnanolone sulfate reach the brain (Neuropharmacology 61, 61-68 (2011)). The transport of sulfated analogs is probably mediated by active exchange mechanisms associated with so-called organic anion transport protein (OATP), which is expressed in the cells of brain tissue.
Inhibitors of the NMDA receptor are also some steroid derivatives and in particular reduced derivatives of progesterone. Its neuroprotective properties are also described in the patent literature (US2012/71453 A1, 2012; WO 2009/108804 A and WO 2009/108809).
These drugs act only under specific conditions of certain structural prerequisites (J. Pharmacol. Exp. Ther. 293, 747-754 (2000)). An essential structural requirement is bent shape of the molecule; this requirement is accomplished by derivatives 3alpha, 5beta-configuration of the steroid skeleton, and also to a lesser extent derivatives with 3alpha, 5alpha-configuration. In addition, the activity is dependent on the presence of ionisable groups within a convenient distance from steroid skeleton, i.e. 2 to 8 atoms. This group may be positively or negatively charged. In previously published articles and patent literature was also always mentioned as essential structural element of the acetyl substituent in position 17 of the steroid skeleton. This structural element appears in progesterone, pregnenolone and pregnanolone (Br. J. Pharmacol. 166, 1069-1083 (2012); Steroids 76, 1409-1418, (2011); WO 2009 108 804 and J. Med. Chem. 8, 426-432 (1965)).
The exception is the patent application U.S. Pat. No. 3,132,160 (1964 patent was not granted) on derivatives of androstane with anesthetic and tranquility action. For the described compounds, however, is characteristic an oxygen atom at position C-11, analogously to clinically used pregnanolone analogue—alfaxalone. Due to the fact that the results of biological tests verifying biological properties of these compounds were never disclosed, we consider the activity specified in this patent to be speculative.
Neuroprotective effect of steroid derivatives with a charged substituent at the C-3 also claimed by two patent applications: WO2010003391 (Anionic pregnane compounds, method for Their Producing and Use of Them) and WO2012/110010 (Pregnanolone derivatives substituted in 3alpha-position with the cationic group, Their method of production, usage and pharmaceutical preparation Involving Them). Both documents claim pregnane derivatives (polar substituent at C-20), substituted at C-3 of an anionic or cationic group of the formula

Derivatives claimed in the present application are not pregnanolone derivatives (these compounds do not have a keto group at the C-20). In case that the claimed compound has a carbonyl group, for the structure-activity reasons at position C-17 and thus, these compounds are androstane derivatives. In case of other polar modification, these derivatives have oxygen substituent in a lower oxidation state (ether) than the C-20 keto group.
Removal of polar substituent at position C-20, modifications and substitution of a D-ring at C-17 or C-16 with non-polar or lipophilic substituents, as well as complete removal of the steroidal D-ring, is likely to lead to better solubility of these derivatives in the membrane and a higher affinity to the NMDA receptor, resulting in some cases in multiple reduction of IC50 values in comparison with the reference compound (3alpha, 5beta-pregnanolone sulfate). Our claimed compounds show that a higher degree of inhibition and IC50 values lower than the reference compound can not generally be predicted, since the substitution or modification at the C- or D-ring in combination with the size and composition of the substituent at C-3 is always unique and it is not possible to predict in advance and to suggest structure by an additive approach. The above mentioned claims are illustrated by examples of substances (Table II) with an IC50 value lower than the reference compound.